The ability to isolate well-characterized human induced pluripotent stem cell derived cardiomyocytes (iCM) from mixed cell populations is a fundamental and major unfulfilled goal of cardiac regenerative medicine, disease modeling and drug discovery efforts. The overall goal of this proposal is to develop a clinically- applicable method for high-throughput isolation of purified ventricular iCM, one of the most sought after cell types for translational applications. The isolation strategy is based on immunophenotyping, wherein functional potential, identity, and isolation of clinically-relevant cells are achieved via panels of antibodies that recognize exposed surface markers. Our preliminary studies have identified more than 40 informative cell surface markers for this purpose and we have developed novel monoclonal antibodies to one novel cell surface protein not previously described in the heart. We have already determined that several of these markers are restricted to specific chambers in the heart, indicating they will be highly informative for sorting subtype-specific iCM in vitro. The work will be carried out in two Aims designed to test the hypothesis that antibodies to cell surface proteins can be used to isolate ventricular iCM (Aim 1) and to determine their functional role in ventricular cardiomyogenesis (Aim 2). This work will contribute a new, non-transgene based high-throughput method for isolating live ventricular cardiomyocytes, which is not possible by current methodologies. The significance of this proposal lies with the functional outcomes enabled by the technological developments, as when an informative marker panel for sorting stage and subtype specific cells is developed, this will enable the reproducible isolation of ventricular iCM suitable for drug-discovery, therapeutic, and disease modeling studies.